This AREA project is to develop a Diverging Flow Electroporation (DFE) system for microfludics-mediated gene/oligonucleotides delivery to both stem cells and cancer cells. The DFE platform lies on the size-specific electroporation on cells with unrestricted population. Two specific aims are proposed: (1) design, construct and validate a DFE system;(2) characterize in vitro delivery efficacy and cell viability for oligonucleotides (ODN G3139 and miR29b) and report genes (pGFP and pSEAP) for cancer and stem cells, respectively. Having cells sequentially passing a focused electric field zone, each of them will receive size-specific electric pulses. It works as many single cell electroporation (SCE) trials are performed in parallel and continuously. This would eliminate the large variations of the pulse strength received by different cells when electroporated in a large population (i.e., bulk electroporation, BE). Therefore, it will not only greatly improve DNA or oligonucleotides delivery efficacy and cell viability, but also facilitate the discovery of the cellular uptake dynamics and simplify the cell-type specific, and cell-population dependent protocols. It may close or largely reduce the gap between SCE for fundamental mechanism study and BE for therapeutic applications. This is particularly valuable for cell systems which either require multiple tasks with limited cell source (e.g., primary cells) or demand efficient treatment with high cell throughput (e.g., stem cells). If successful, it will benefit the life science and biomedical community where a safe and effective non-viral gene delivery approach is urgently needed. The proposed research topics will help strengthen the collaborations between the Institute for Micromanufacturing (IfM) and the Center for Biomedical Engineering and Rehabilitation Science (CBERS) at Louisiana Tech University and provide unique interdisciplinary research training to their students. The success of this project will also help establish the PI's leadership in micro/nanoengineering cell therapy and broaden sustained participation of him and his colleagues in NIH programs since LA Tech has not been a major recipient of NIH support. It will also benefit the students by inspiring their research passions to further explore health-related topics in advanced study. PUBLIC HEALTH RELEVANCE: A Diverging Flow Electroporation (DFE) system will be developed for microfludics-mediated gene/oligonucleotides delivery to both stem cells and cancer cells. This new technology will provide a size-specific electroporation on every cell, so as to achieve high delivery efficacy, high cell viability, high cell throughput, and quick protocol identification. If successful, this might yield tremendous medical benefits for diagnosing, treating and preventing diseases.